The Effects of Zn2+ on the Binding of the BDNF Prodomain
with SorCS2 on Neuronal Function and Structure:
Implications for Learning and Memory
Caroline Pennacchio
Briarcliff Manor High School
The Effects of Zn2+ on the Binding of the BDNF Prodomain with SorCS2 on Neuronal Function and Structure:Implications for Learning and Memory Caroline Pennacchio Briarcliff High School, Briarcliff Manor, NY
Neurotrophic factors are types of ligands found throughout the brain and are secreted by neurons to other neurons. They are in charge of regulating cell proliferation, differentiation, axon and dendrite growth, synaptogenesis, and synaptic function and plasticity. The four main ligands that interact in the brain are BDNF, NGF, NT-3 and NT-4/5. Although many times the functions of proneurotrophins in the brain are ignored, pro-neurotrophins have the ability to interact with p75 neurotrophin with high affinity, which demonstrates that the pro-region could be paramount to p75 signaling despite the ability of mature neurotrophins such as NGF to bind with p75. Furthermore, proBDNF acts in vivo as a biologically active factor regulating hippocampal structure, and synaptic transmission, and plasticity, which mature BDNF does not impact. Small changes in the human BDNF gene can lead to a Val66Met substitution within the prodomain. This single-nucleotide polymorphism (SNP) of BDNF has been linked with depression, memory alterations, and anxiety disorders and substitution induces structural changes in the protein.This study was conducted in order to determine the consequences of growth contraction and synapse elimination. Previously the impacts of the Val66Met polymorphism of BDNF on prodomain structure were determined to impact the structure and function of the protein produced altering growth cone retraction. Previously, a 28% binding differential of the Val and Met isoforms was seen. Therefore, the binding of the two isoforms of prodomain was examined with SorCS2 in the presence of Zn2+. However, the binding of the Met and Val isoforms appeared to be the same which should not have occurred as binding differences were seen previously. There are plans in place for the continuation of the research on this change to help reverse the depressional impacts of these SNPs.
Review of Literature
The brain is divided into the frontal, parietal, temporal and occipital lobes, each of which
is responsible for different functions in the brain. In terms of memory, the frontal lobe works to
coordinate transient working memory and working memory. The hippocampus, a small, pea-
sized region in the center of the brain, works to maintain plasticity through the use of negative
and positive remodeling which leads to memory formation. Furthermore, different molecules
participate in neural connections leading to the production of Brain-derived neurotrophic factor
(BDNF) which increases hippocampal neurogenesis, and alters neuronal function.
Neurotrophic factors are types of ligands found throughout the brain and are secreted by
neurons to other neurons. In charge of regulating cell proliferation, differentiation, axon and
dendrite growth, synaptogenesis, and synaptic function and plasticity, (Reichardt, 2006, Lu, B,
Pang, PT, Woo. N.H., 2005, Minichiello L, 2009) neurons function interdependently to send
signals along an axon to the cell body and axon of the neighboring neurons. The four main
neurotrophic ligands in the brain are BDNF, NGF, NT-3 and NT-4/5. Each of these ligands has
the ability to bind to p75 along with a specific neurotrophin receptor known as a Trk receptor.
NGF binds with TrkA, BDNF and NT-4/5 bind with Trk B, and NT-3 binds with TrkC
(Bothwell, 1991). However, BDNF and NT-4/5 may be referred to as “functionally redundant,”
because of their identical Trk compound binding patterns (Chao 2003).
Secreted in an activity and calcium ion dependent manner, BDNF may be involved in
controlling synaptic transmission and long-term synaptic plasticity because of its ability to
promote neurogenesis. Additionally, the secretion of BDNF as a response to activity and calcium
ion production represents an important mechanism underlying local and synapse-specific BDNF
modulation (Lu, 2003). Changes occur in genes in the form of single nucleotide polymorphisms (SNP). A SNP of BDNF results in a Val66Met substitution in the pro domain region, and has been linked with depression, memory alterations, and anxiety disorders in humans because of the internal disordering of the prodomain and the Val66Met substitution which induce structural changes
(Anastasia et al., 2013). While BDNF is responsible for many of the main functions of neurons,
NT-4/5 also activates TrkB which in turn activates the tyrosine kinase compound activation of
PI3K (phosphatidylinositol 3kinase) (Chao, 2003).
There are two main types of neurotrophins: proneurotrophins and mature neurotrophins.
While pro-neurotrophins consist of about 270 amino acids, with the pro-region consisting of approximately 120 amino acids (Seidah et al., 1996), mature neurotrophins are different from proneurotrophins in both structure and function. First synthesized as precursors, proneurotrophins are then proteolytically cleaved in trans-Golgi by furin or secretory granules by pro-protein contervases. This results in the formation of mature neurotrophins (the “pro” region has been removed) (Seidah el al., 1996) and the formation of the mature domain (proBDNF splits into BDNF and the prodomain). Pro and mature neurotrophins have opposite functional effects resulting from their diverse interactions with TrkA and p75 receptors. However, the extracellular cleavages that are created during the formation of the mature domain demonstrate how synaptic functions of neurotrophins can be controlled (Lu, 2003). Although many times the functions of proneurotrophins in the brain are ignored, pro-neurotrophins have the ability to interact with p75 neurotrophin with high affinity, which demonstrates that the pro-region could be paramount to p75 signaling (Lee et al,. 2001) despite the ability of mature neurotrophins such as NGF to bind with p75. Furthermore, proBDNF acts in vivo as a biologically active factor regulating hippocampal structure, and synaptic transmission, and plasticity, which mature BDNF impacts differently (Yang et al., 2014). The ability of sortilin to recognize the prodomains or the
3 proneurotrophins: proNGF, proBDNF and pro NT-3, allows for the formation of a co-receptor complex of p75, sortilin and the proneurotrophin. This demonstrates proneurotrophin induced apoptotic signaling at lower ligand concentrations (Nykjaer et al. 2004; Teng et al. 2005; Jansen et al. 2007; Willnow et al. 2008; Yano et al. 2009).
SorCS2 is another receptor and gene found within the nervous system. A member of the sortilin family, this receptor binds with a variety of ligands to mediate neuronal activities.
Additionally, deficits caused by lack of SorCS2 receptor can result in problems for the organism, as sortilin, a biologically important neurotrophin receptor, targets the pro-domain of proNGF with high affinity (Nykjaer, et al, 2004). However, sortilin deficiency does not affect developmentally regulated apoptosis of sympathetic neurons, or prevent age-dependent degeneration (Jansen, et al., 2007).
This study was conducted in order to determine the consequences of growth contraction and synapse elimination. Previously the impacts of the Val66Met polymorphism of BDNF on prodomain structure were determined to impact the structure and function of the protein produced altering growth cone retraction.
Previously, a 28% binding differential of the Val and Met isoforms was seen (Anastasia,
2013).
Although using Biocore assay was a potential approach, the biology of the prodomain molecule would have most likely caused the protein to stick to the Biocore instead of binding with both Zn2+ and the receptor SorCS2 when they were present. The Biocore also would have made any binding difficult to see. Therefore, using Co-IP with was selected instead.
Research Question(s) and Hypotheses:
Q1: How does SorCS2 interact with the prodomain?
Q2: How does the met SNP influence prodomain
effects?
H0 Null Hypothesis: They interact differently
H1 Refined Hypothesis: The Met prodomain will have more pronounced effects than the Val
prodomain.
In the presence of Zinc: Val will stay constant and bind with SorCS2 a small amount of binding
product will be formed. Met will increase and bind with SorCS2 a large amount of binding
product will be formed
H2 Protein Purification Hypothesis: If you knock out SorCS2 receptor the Met prodomain will
not have any effect.
Binding of Prodomain with SorCS2 A previous study (Anastasia, 2013), showed that
Met66 prodomain binds differentially and stronger
with SorCS2 than the Val66 prodomain (through
quantitative analysis). It is possible that the
differences in function and structures of the
different isoform-containing prodomains have
resulted from this binding difference. As a result of
the conclusions of this study, it was decided that the
impact of Zn2+ (an ion commonly found in the
Figure A post-synaptic cleft) on this interaction should be
examined more thoroughly.
Testing Prodomain Binding to Avidin Agarose beads Test Trial Using Co-IP
Conditions: Prodomain, Prodomain + Zn2+, Prodomain + Zn2+ + NaCl (both pre and post beads samples for each condition)
Co-IP was performed using Val66 prodomain samples.
Following SDS-Page, the membrane was incubated with
proBDNF Gene Copeoia antibody (1:10,000 dilution)
followed by HRP.
Figure 1.
Results: The goal of this trial was to determine nonspecific binding. The question: Are there
prodomain alterations dependent on SorCS2 binding with Zn2+? The goal was to identify
conditions where there is not much non-specific binding. As evidenced in figure 1, Prodomain
binds with Avidin Agarose beads in the presence and absence of Zn2+ and NaCl. Additionally,
this demonstrates that the prodomain or prodomain complex in binding with the avidin beads. Prodomain/Zn2+ Complex Binding to SorCS2 using Co-IP/ Striping and Probing For Biotin and SorCS2 SorCS2 was biotinylated. Co-IP was performed with both Val
and Met samples in the presence and absence of both SorCS2
and Zn2+. Following SDS- Page, the membrane was incubated
with Ab SorCS2 (at a 1:1000 dilution) followed by Ab Shp
HRP (at a 1:5000 dilution). A staining kit was SorCS2 used to detect biotinylated protein (Vectastain
Biotin ABC Kit).
Conditions: Val/Met w/o SorCS2 in the absence
Prodomain of Zn2+, Val/Met with SorCS2 in the absence of Zn2+, Val/Met w/o SorCS2 in the presence of Zn2+, Val/Met with SorCS2 in the presence of Zn2+
Results: As evidenced in figure 2, the biotinylation of SorCS2 was successful and did not
interfere with the presence of Prodomain. There was a 10:1 ratio and 1/10 binding which
suggests that SorCS2 could bind to the prodomain. Both the Val and Met isoforms of BDNF
prodomain were present when probed for.
Figure 2. *One membrane with glutaldehyde. SorCS2 cannot be detected
Prodomain/Zn2+/SorCS2 Complex Binding with SorCS2 and Prodomain Using Co-IP Co-IP was performed followed by SDS-Page. All Co-IP
washed were performed with Zn2+. Following SDS-Page
the blot was probed for both SorCS2 and prodomain using
SorCS2 antibody (1:10 dilution) and proBDNF Gene
Copeoia antibody (1:10,000 dilution) respectively, followed
by HRP. Conditions: Val/Met w/o SorCS2 in the absence of Zn2+, Val + Zn2+, Met + Zn2+. Val/Zn2+/SorCS2, Met + SorCS2 SorCS2. Val/Zn2+/SorCS2
Results: More nonspecific binding with the Prodomain avidin agarose beads occurred than was detected Figure 3. in the past. Additionally, this aggregation was
greater in the Met than in the Val isoform. Prodomain/Zn2+/SorCS2 Complex Binding with SorCS2 Using Co-IP Figure 4.
Co-IP was performed followed by SDS-Page. All Co-IP
washed were performed with Zn2+. Following SDS-Page
the blot was probed for SorCS2 using SorCS2 antibody
(1:10 dilution) followed by HRP.
Results: There was a lack of difference in the amount of binding of the Val and Met isoforms. The amount of binding in Conditions: Val/Met w/o Zn2+ and w/o SorCS2, Val the presence of SorCS2 was slightly + Zn2+, Met + Zn2+, Val +SorCS2, Met + SorCS2, greater. Val/Zn2+/SorCS2, Met/Zn2+/SorCS2 SorCS2 Binding to Agarose Avidin Beads using Co-IP Co-IP was performed followed by SDS-Page. Following
SDSPage, the blot was probed for SorCS2 using SorCS2
antibody (1:10 dilution) followed by HRP.
Conditions: SorCS2 and both 4° and -80°, includes both supernatant and bound SorCS2
Results: SorCS2 stored in both 4° and -80° demonstrates the
ability to bind with Agarose Avidin beads. The ability of the
SorCS2 stored at 4° to bind with the beads is slightly better
however. A proportion was precipitated.
Figure 5. Prodomain/Zn2+ Complex Binding to SorCS2
Following 15 minutes of incubation, Co-IP was performed
followed by SDS-Page. Following SDS-Page, the blot was
probed for prodomain (using proBDNF antibody at a
1:10,000 dilution from Gene Copoeia followed by HRP) and
SorCS2 (using homemade SorCS2 antibody in a 1:10 dilution
followed by HRP). Figure 6. Conditions: Met/Val + Zn2+, Met/Val + Zn2+ + SorCS2, Control: SorCS2
Results: There was a lot of nonspecific binding of SorCS2 to the avidin agarose beads. Therefore
because the conditions are not right, the hypotheses cannot be proven nor disproven. Discussion/Conclusion:
Figure 1 demonstrated the conditions in which there is not much nonspecific binding of
prodomain to the beads. This is especially important because the structure of the
prodomain/SorCS2 complex and prodomain itself makes it a molecule that readily and easily
binds with other receptors. A problem with the procedure followed for figure 1 is the possibility
that the fast dissociation resulted in more non-specific binding and less specific binding because
of the long washes. These could have occupied the binding sites of the molecule that might have
otherwise bound with the receptor complex. The two main isoforms of prodomain (Met and Val)
which were explored throughout this investigation result in the induction of different structures
of the prodomain molecule and therefore inspire different functions in the molecule. The Met
and Val prodomains interact with SorCS2 different as has been seen in previous studies which
affirms my first hypothesis.
However, although the Met prodomain should have demonstrated more pronounced
binding with SorCS2, this was neither proven nor disproved. Although there was a 10:1 ratio
between the prodomain and SorCS2 as shown in figure 2 and a 1/10 binding was confirmed
which suggests that SorCS2 may induce prodomain binding no control existed. Possible causes
of this include problems in the biotinylation of the SorCS2 or that these results could be the best
possible results at the base amount of SorCS2 used.
In figure 3, there was also more nonspecific binding coming down with the beads in the
Met than the Val which could have resulted in these large aggregates which pellet inconsistently.
The main question is as to why this failure to stick occurred. In figure 4 there was also not a lot of binding although the SorCS2 binding was slightly greater. However, the binding of the Met and Val isoforms appeared to be the same which should not have occurred per the results of Anastasia, 2013.
As a result of the problems with the previous assays, a SorCS2 assay was run (as seen in figure 5) to determine the ability to precipitate SorCS2. The assay was successful as a proportion was precipitated and proved the ability of the SorCS2 to bind with the beads.
In figure 6, all of the accumulation occurred on the beads. A lot of non-specific binding occurred as well. This demonstrated that the conditions of the experiment were not right.
Following these trials, a similar set will be rerun. SorCS2 will be biotinylated, the amount of biotin binding with the SorCS2 will be assayed and the impact of SorCS2 on prodomain binding with Zn2+ and without Zn2+ will be assayed with an examination in the differences in prodomain binding of the Met and Val isoforms. Hopefully this change can allow research to reverse the depressional impacts of these SNPs.
Bibliography
Anastasia, A., Deinhardt, K., Chao, M. V., Will, N. E., Irmady, K., Lee, F. S., ... & Bracken, C.
(2013). Val66Met polymorphism of BDNF alters prodomain structure to induce neuronal growth cone retraction. Nature communications, 4..
Beattie, M. S., Harrington, A. W., Lee, R., Kim, J. Y., Boyce, S. L., Longo, F. M., ... & Yoon, S.
O. (2002). ProNGF induces p75-mediated death of oligodendrocytes following spinal cord injury. Neuron, 36(3), 375-386.
Bothwell, M. (2014). NGF, BDNF, NT3, and NT4. In Neurotrophic Factors (pp. 3-15). Springer
Berlin Heidelberg.
Bothwell, M. (1995). Functional interactions of neurotrophins and neurotrophin receptors.
Annual review of neuroscience, 18(1), 223-253.
Bothwell, M. (1991). Keeping track of neurotrophin receptors. Cell, 65(6), 915-918.
Chao, M. V. (2003). Neurotrophins and their receptors: a convergence point for many signalling pathways. Nature Reviews Neuroscience, 4(4), 299-309.
Deinhardt, K., & Chao, M. V. (2014). Trk Receptors. In Neurotrophic Factors(pp. 103-119).
Springer Berlin Heidelberg.
Glerup, S., Nykjaer, A., & Vaegter, C. B. (2014). Sortilins in Neurotrophic Factor Signaling. In
Neurotrophic Factors (pp. 165-189). Springer Berlin Heidelberg.
Hasan, W., Pedchenko, T., Krizsan Agbas, D., Baum, L., & Smith, P. G. (2003). Sympathetic neurons synthesize and secrete pro nerve growth factor protein. Journal of neurobiology, 57(1),
38-53.
Hempstead, B. L. (2014). Deciphering Proneurotrophin Actions. In Neurotrophic Factors (pp.
17-32). Springer Berlin Heidelberg.
Hempstead, B. L. (2002). The many faces of p75< sup> NTR. Current opinion in neurobiology, 12(3), 260-267.
Hermey, G., Keat, S. J., Madsen, P., Jacobsen, C., Petersen, C. M., & Gliemann, J. (2003).
Characterization of sorCS1, an alternatively spliced receptor with completely different cytoplasmic domains that mediate different trafficking in cells. Journal of Biological Chemistry,
278(9), 7390-7396.
Jacobsen, L., Madsen, P., Jacobsen, C., Nielsen, M. S., Gliemann, J., & Petersen, C. M. (2001).
Activation and functional characterization of the mosaic receptor SorLA/LR11. Journal of
Biological Chemistry, 276(25), 22788-22796.
Jansen, P., Giehl, K., Nyengaard, J. R., Teng, K., Lioubinski, O., Sjoegaard, S. S., ... & Nykjaer,
A. (2007). Roles for the pro-neurotrophin receptor sortilin in neuronal development, aging and brain injury. Nature neuroscience, 10(11), 1449-1457.
Klein, A. B., Williamson, R., Santini, M. A., Clemmensen, C., Ettrup, A., Rios, M., ... & Aznar,
S. (2011). Blood BDNF concentrations reflect brain-tissue BDNF levels across species. The
International Journal of Neuropsychopharmacology, 14(03), 347-353.
Lee, R., Kermani, P., Teng, K. K., & Hempstead, B. L. (2001). Regulation of cell survival by secreted proneurotrophins. Science, 294(5548), 1945-1948. Lu, Bai (2014). Pro-Region of Neurotrophins: Role in Synaptic Modulation. In Neurotrophic
Factors Springer Berlin Heidelberg.
Lu, B. (2003). BDNF and activity-dependent synaptic modulation. Learning & Memory, 10(2),
86-98.
Lu, B. (2003). Pro-region of neurotrophins: role in synaptic modulation. Neuron, 39(5), 735-738.
Mazella, J., Zsürger, N., Navarro, V., Chabry, J., Kaghad, M., Caput, D., ... & Vincent, J. P.
(1998). The 100-kDa neurotensin receptor is gp95/sortilin, a non-G-protein-coupled receptor.
Journal of Biological Chemistry, 273(41), 26273-26276.
Nykjaer, A., Lee, R., Teng, K. K., Jansen, P., Madsen, P., Nielsen, M. S., ... & Petersen, C. M.
(2004). Sortilin is essential for proNGF-induced neuronal cell death. Nature, 427(6977), 843848.
Park, H., & Poo, M. M. (2012). Neurotrophin regulation of neural circuit development and function. Nature Reviews Neuroscience, 14(1), 7-23..
Petersen, C. M., Nielsen, M. S., Jacobsen, C., Tauris, J., Jacobsen, L., Gliemann, J., ... &
Madsen, P. (1999). Propeptide cleavage conditions sortilin/neurotensin receptor 3 for ligand binding. The EMBO journal, 18(3), 595-604.
Rodriguez-Tebar, A., Dechant, G., & Barde, Y. A. (1990). Binding of brain-derived neurotrophic factor to the nerve growth factor receptor. Neuron, 4(4), 487-492.
Rodriguez-Tebar, A., Dechant, G., Götz, R., & Barde, Y. A. (1992). Binding of neurotrophin-3 to its neuronal receptors and interactions with nerve growth factor and brain-derived neurotrophic factor. The EMBO journal, 11(3), 917.
Ryden, M., Murray-Rust, J., Glass, D., Ilag, L. L., Trupp, M., Yancopoulos, G. D., ... & Ibanez,
C. F. (1995). Functional analysis of mutant neurotrophins deficient in low-affinity binding reveals a role for p75LNGFR in NT-4 signalling. The EMBO journal, 14(9), 1979.
Sarret, P., Krzywkowski, P., Segal, L., Nielsen, M. S., Petersen, C. M., Mazella, J., ... &
Beaudet, A. (2003). Distribution of NTS3 receptor/sortilin mRNA and protein in the rat central nervous system. Journal of Comparative Neurology, 461(4), 483-505.
Seidah, N. G., Benjannet, S., Pareek, S., Chrétien, M., & Murphy, R. A. (1996). Cellular processing of the neurotrophin precursors of NT3 and BDNF by the mammalian proprotein convertases. FEBS letters, 379(3), 247-250.
Teng, H. K., Teng, K. K., Lee, R., Wright, S., Tevar, S., Almeida, R. D., ... & Hempstead, B. L.
(2005). ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin. The journal of neuroscience, 25(22), 5455-5463.
Wehrman, T., He, X., Raab, B., Dukipatti, A., Blau, H., & Garcia, K. C. (2007). Structural and mechanistic insights into nerve growth factor interactions with the TrkA and p75 receptors.
Neuron, 53(1), 25-38.
Willnow, T. E., Petersen, C. M., & Nykjaer, A. (2008). VPS10P-domain receptors—regulators of neuronal viability and function. Nature Reviews Neuroscience, 9(12), 899-909.
Yang, J., Harte-Hargrove, L. C., Siao, C. J., Marinic, T., Clarke, R., Ma, Q., ... & Hempstead, B.
L. (2014). proBDNF Negatively Regulates Neuronal Remodeling, Synaptic Transmission, and
Synaptic Plasticity in Hippocampus. Cell reports, 7(3), 796-806.
Yano, H., Torkin, R., Martin, L. A., Chao, M. V., & Teng, K. K. (2009). Proneurotrophin-3 is a neuronal apoptotic ligand: evidence for retrograde-directed cell killing. The Journal of
Neuroscience, 29(47), 14790-14802.